VEGF acts on vascular endothelial cells to increase cell permeability and to stimulate cell replication, events which occur during ovulation and luteinization of the follicle. This study evaluated the effect of in vivo gonadotropin treatment given as the midcycle ovulatory surge on macaque granulosa cell VEGF production. In Expt. 1, monkeys received urinary gonadotropins [30 IU FSH bid (Metrodin, Ares Serono) for 6 days followed by 30 IU FSH+ 30 IU LH bid (Pergonal, Serono) for 2-3 days] to promote multiple follicular development, followed by no ovulatory stimulus (n=6) or the standard ovulatory stimulus (1000 IU hCG, Profasi, Ares Serono; n=6). In Expt. 2, monkeys received a follicular stimulation protocol of recombinant (r)-hFSH (30 IU, Serono, bid) followed by a bolus of 100 or 1000 IU of r-hCG (Ares Serono) or 2500 IU of r-hFSH (n=5 /group). Nonluteinized granulosa cells (i.e., no ovulatory stimulus) and luteinized granulosa cells (i.e., bolus of hCG or FSH) were collected the day after the last treatment. Cells were incubated for 24 hr in the presence or absence of 100 ng/ml of hCG and media assayed for VEGF by ELISA (R&D Systems, Minneapolis, MN). In Expt. 1, luteinized granulosa cells from hCG-treated monkeys produced greater (P<0.05) levels of VEGF (mean q SEM; 899 q 471 pg/ml) than nonluteinized granulosa cells (111 q 26). Further, while in vitro treatment with hCG tended (P<0.067) to increase VEGF (1162 q 621) levels produced by luteinized granulosa cells, nonluteinized granulosa cell VEGF production increased (P<0.05) 4-fold (470 q 147) in response to hCG. In Expt. 2, hCG (100 and 1000 IU) and hFSH (2500 IU) in vivo were equally effective in elevating basal VEGF levels (734 q 119, 799 q 167 and 675 q 103, respectively) to values similar to those for luteinized granulosa cells in Expt. 1. In vitro exposure to hCG increased (P<0.05) VEGF production by the granulosa cells from the 100 IU hCG (1354 q1 46), 1000 IU hCG (973 q 217) and 2500 IU r-hFSH (1230 q 382) groups. These experiments provide the first direct evidence that the midcycle surge of gonadotropins stimulates VEGF production by granulosa cells in the preovulatory follicle of primates. Moreover, the evidence suggests that a bolus of FSH, as well as LH/CG, is capable of promoting VEGF production by granulosa cells.